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Open Access Green as soon as Postprint is submitted to ZB.
His164 regulates accessibility to the active site in fungal 17β-hydroxysteroid dehydrogenase.
Biochimie 89, 63-71 (2007)
17ß-Hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17ß-HSDcl) is an NADPH-dependent member of the short-chain dehydrogenase/ reductase superfamily. To study the catalytic properties of this enzyme, we prepared several specific mutations of 17ß-HSDcl (Tyr167Phe, His164Trp/Gly, Tyr212Ala). Wild-type 17ß-HSDcl and the 17ß-HSDcl mutants were evaluated by chromatographic, kinetic and thermodynamic means. The Tyr167Phe mutation resulted in a complete loss of enzyme activity, while substitution of His164 with Trp and Gly both resulted in higher specificity number (V/K) for the steroid substrates, which are mainly a consequence of easier accessibility of steroid substrates to the active-site hollow under optimized conditions. The Tyr212Ala mutant showed increased activity in the oxidative direction, which appears to be a consequence of increased NADPH dissociation. The kinetic characterizations and thermodynamic analyses also suggest that His164 and Tyr212 in 17ß-HSDcl have a role in the opening and closing of the active site of this enzyme and in the discrimination between oxidized and reduced coenzyme.
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Publication type
Article: Journal article
Document type
Scientific Article
Keywords
17ß-Hydroxysteroid dehydrogenase; Short-chain dehydrogenase/reductase; Site-directed mutagenesis; Kinetic characterization; Thermodynamic analysis
ISSN (print) / ISBN
0300-9084
e-ISSN
1638-6183
Journal
Biochimie
Quellenangaben
Volume: 89,
Issue: 1,
Pages: 63-71
Publisher
Elsevier
Non-patent literature
Publications
Reviewing status
Peer reviewed
Institute(s)
Molekulare Endokrinologie und Metabolismus (MEM)